Effect Of Baicalin On Apoptosis Of Porcine Peritoneal Mesothelial Cells Induced By Glaesserella Parasuis And Its Molecular Mechanism

In this study,transcriptome sequencing was performed on porcine peritoneal mesothelial cells(PPMC)infected with Glaesserella parasuis(GPS)to investigate the molecular mechanism of damage to PPMC.The effects of baicalin on apoptosis and the PKC-MAPK signaling pathway of PPMC cells induced by GPS were also studied.1.Transcriptome sequencing analysis of GPS-infected PPMC.Negative control group and GPS infection model group were set up in the experiment,with 3 replicates in each group.According to the previous study of our group,the MOI was determined as 100:1.After 12h of co-culture,RNA samples were extracted and transcriptome sequencing was performed.12 selected differential genes were verified by real-time quantitative PCR.The results showed that there were 779 differentially expressed genes(P<0.05),in which 253 genes were up-regulated and 526 genes were down-regulated.Among the 12 genes selected and verified,10 genes showed similar expression levels with RNA-seq data.The main inflammatory pathways and MAPK pathway were activated after GPS infection of PPMC,showing the cellular inflammatory response and affecting the cell growth related pathways.2.Effect of baicalin on apoptosis in GPS-infected PPMC.Five experimental groups,including negative control group,10~7 CFU/m L GPS model group,GPS+baicalin treatment group(25,50 and 100μg/m L,respectively),were set in the experiment.The initial number of cells was 10~5/m L,and the concentration shown by GPS was the final concentration.PPMC were pretreated with drugs for 1h,and then co-cultured with bacteria for 12h.The m RNA expression levels of apoptosis genes Bax,Bcl-xl and C-myc were determined by real-time fluorescence quantitative PCR.The expression of cleaved caspase-3 protein was determined by Western blot.Cell apoptosis was detected by flow cytometry.The results showed that after PPMC was infected with GPS,the levels of Bax,Bcl-xl and C-myc were significantly increased,the expression of cleaved caspase-3 protein was significantly increased,and the cell apoptosis rate was significantly increased.25μg/m L baicalin could significantly down-regulate the expression of Bax gene(P<0.05),significantly decreased the apoptotic rate of infected cells(P<0.05).Baicalin at 50μg/m L could significantly down-regulate the expression of Bax gene(P<0.05),Bcl-XL gene(P<0.05),and C-myc gene(P<0.01),significantly decreased cleaved caspase-3 protein expression(P<0.01)and reduced the apoptotic rate of infected cells.Baicalin at 100μg/m L could significantly down-regulate the expression of Bcl-XL gene(P<0.01)and C-myc gene(P<0.05),significantly decreased cleaved caspase-3 protein expression(P<0.01)and reduced the apoptotic rate of infected cells(P<0.01).3.Effects of baicalin on PKC-MAPK signaling pathway in GPS-infected PPMC.Same experimental groups were used as above.PPMC were pretreated with drugs for1h and then co-cultured with bacteria for 12 h.The expression levels of PKC-MAPK signaling pathway related proteins(PKC-α,p-PKC-α,PKC-δ,p-PKC-δ,ERK,p-ERK,JNK,p-JNK,p38,p-p38,and cleaved caspase-3)were determined by Western blot.The m RNA expression levels of nuclear transcription factors AP-1(c-fos and c-jun)were determined by Q-RT PCR.The results showed that the expression of PKC-MAPK signaling pathway proteins(p-PKC-α,p-PKC-δ,p-ERK,p-JNK and p-p38)and the m RNA expression levels of nuclear transcription factors AP-1(c-fos and c-jun)were significantly up-regulated in GPS-infected PPMC(P<0.01).Baicalin at 50 and 100μg/m L could significantly down-regulated the expression levels of p-PKC-α,p-PKC-δ,p-p38,p-ERK,and p-JNK(P<0.01).25μg/m L baicalin could significantly down-regulated the gene expression of nuclear transcription factors AP-1(c-fos and c-jun)(P<0.01).Baicalin at 50 and 100μg/m L could significantly down-regulate the gene expression of apoptotic nuclear transcription factor AP-1(P<0.01).In summary,GPS infection could cause apoptosis and activate the PKC-MAPK signaling pathway in PPMC.Baicalin may play an anti-apoptotic role by inhibiting the activation of PKC-MAPK signaling pathway in PPMC.This study further clarified the pathogenic mechanism of GPS and the molecular mechanism of baicalin in the prevention and treatment of GPS.